This project studies the physiology and development of calcium regulation during synaptic activity in the dendrites and dendritic spines of central nervous system neurons. Previously, analytical and structural work using a new type of hippocampal slice culture had characterized a subset of endoplasmic reticulum as the major calcium- sequestering organelle in dendrites of CA3 neurons in the minutes following the afferent synaptic activity. We have now established that these calcium-buffering organelles are very similar, if not identical, to those which at other times act as calcium storage and release organelles. It is found that these organelles function as calcium sequestration sites within a time frame of <1-15 minutes, while mitochondrial calcium sequestration operates in parallel with ER uptake only over the first minute following synaptic activity. Similar evidence for robust mitochondrial calcium sequestration has been obtained from a new study on isolated neurons from frog sympathetic ganglia. Previously we had shown that estradiol doubles dendritic spine density in cultures of CA1 hippocampal neurons, thus providing an attractive explanation for the recent discovery that estrogen affects cognition and plasticity in adult organisms. New experiments using kinase inhibitors, antisense oligonucleotides, and specific antibodies in combination with high-resolution confocal microscopy now show that the cAMP response element binding protein (CREB) is necessarily phosphorylated via cAMP- dependent protein kinase A, and CREB binding protein (CBP) is recruited, prior to spine development. This indicates that genome activation to transcribe spine proteins may be required for new spine development. In parallel experiments on the indirect effects of estradiol, it was found that this hormone lowers glutamic acid decarboxylase (GAD) in inhibitory hippocampal interneurons, thus decreasing the amount and availability of the neurotransmitter GABA. This effectively changes the balance of excitation/inhibition in pyramidal neurons, increasing intracellular calcium which may in turn cause phosphorylation of CREB and synthesis of new spines. The findings suggest that the indirect effects of estradiol can provide a powerful yet transient way to control excitatory activity and morphological plasticity.